WO2019050813A1 - Polysaccharides antipneumococciques et leur utilisation dans des conjugués immunogènes polysaccharide-protéine porteuse - Google Patents

Polysaccharides antipneumococciques et leur utilisation dans des conjugués immunogènes polysaccharide-protéine porteuse Download PDF

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WO2019050813A1
WO2019050813A1 PCT/US2018/049305 US2018049305W WO2019050813A1 WO 2019050813 A1 WO2019050813 A1 WO 2019050813A1 US 2018049305 W US2018049305 W US 2018049305W WO 2019050813 A1 WO2019050813 A1 WO 2019050813A1
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polysaccharide
kda
protein
immunogenic composition
glycerol
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PCT/US2018/049305
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English (en)
Inventor
Richard J. PORAMBO
Chitrananda Abeygunawardana
Luwy Kavuka MUSEY
Michael J. KOSINSKI
Yadong Adam CUI
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Merck Sharp & Dohme Corp.
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Priority to BR112020004471A priority Critical patent/BR112020004471A8/pt
Priority to CN201880058174.XA priority patent/CN111093649B/zh
Priority to RU2020112309A priority patent/RU2801288C2/ru
Priority to KR1020207009612A priority patent/KR20200051002A/ko
Application filed by Merck Sharp & Dohme Corp. filed Critical Merck Sharp & Dohme Corp.
Priority to AU2018328035A priority patent/AU2018328035B2/en
Priority to JP2020513531A priority patent/JP7293201B2/ja
Priority to CN202311046367.2A priority patent/CN116898959A/zh
Priority to US16/645,023 priority patent/US11395849B2/en
Priority to CA3074703A priority patent/CA3074703A1/fr
Priority to EP18852920.0A priority patent/EP3678653A4/fr
Priority to MX2020002557A priority patent/MX2020002557A/es
Publication of WO2019050813A1 publication Critical patent/WO2019050813A1/fr
Priority to US17/848,466 priority patent/US11759510B2/en
Priority to US17/848,468 priority patent/US11759511B2/en
Priority to AU2024201470A priority patent/AU2024201470A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • C—CHEMISTRY; METALLURGY
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    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • A—HUMAN NECESSITIES
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
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    • A61P37/00—Drugs for immunological or allergic disorders
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    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
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    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
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    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
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    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin

Definitions

  • the present invention provides purified capsular polysaccharides from Streptococcus pneumoniae serotype 16F, and polysaccharide-protein conjugates having polysaccharides from this serotype. Polysaccharide-protein conjugates from this serotype may be included in multivalent pneumococcal conjugate vaccines.
  • Streptococcus pneumoniae is a significant cause of serious disease world-wide.
  • the Centers for Disease Control and Prevention estimated there were 3,000 cases of pneumococcal meningitis, 50,000 cases of pneumococcal bacteremia, 7,000,000 cases of pneumococcal otitis media and 500,000 cases of pneumococcal pneumonia annually in the United States.
  • CDC Centers for Disease Control and Prevention
  • the complications of these diseases can be significant with some studies reporting up to 8% mortality and 25% neurologic sequelae with pneumococcal meningitis. See Arditi et al, 1998, Pediatrics 102: 1087-97.
  • the multivalent pneumococcal polysaccharide vaccines that have been licensed for many years have proved invaluable in preventing pneumococcal disease in adults, particularly, the elderly and those at high-risk.
  • infants and young children respond poorly to unconjugated pneumococcal polysaccharides.
  • Bacterial polysaccharides are T-cell- independent immunogens, eliciting weak or no response in infants. Chemical conjugation of a bacterial polysaccharide immunogen to a carrier protein converts the immune response to a T- cell-dependent one in infants.
  • Diphtheria toxoid (DTx, a chemically detoxified version of DT) and CRM197 have been described as carrier proteins for bacterial polysaccharide immunogens due to the presence of T-cell-stimulating epitopes in their amino acid sequences.
  • the pneumococcal conjugate vaccine, Prevnar ® containing the 7 most frequently isolated serotypes (4, 6B, 9V, 14, 18C, 19F and 23F) causing invasive pneumococcal disease in young children and infants at the time, was first licensed in the United States in February 2000. Following universal use of Prevnar" in the United States, there has been a significant reduction in invasive pneumococcal disease in children due to the serotypes present in Prevnar ® . See Centers for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep 2005, 54(36): 893- 7.
  • Prevnar 13 ® is a 13 -valent pneumococcal polysaccharide-protein conjugate vaccine including serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. See, e.g., U.S. Patent Application Publication No. US 2006/0228380 Al, Prymula et al, 2006, Lancet 367:740-48 and Kieninger et al, Safety and Immunologic Non-inferiority of 13-valent
  • S. pneumoniae has been categorized into more than ninety serotypes based on the structure of the capsular polysaccharide.
  • a list of known pneumococcal capsular polysaccharide structures is provided in Geno, 2015, Clinical Microbiology Reviews 28:871-899.
  • the current multivalent pneumococcal conjugate vaccines have been effective in reducing the incidence of pneumococcal disease associated with those serotypes present in the vaccines.
  • the prevalence of the pneumococci expressing serotypes not present in the vaccine has been increasing. Accordingly, there is a need to identify and characterize emerging pneumococcal serotypes for inclusion in future vaccines.
  • the present invention provides purified capsular polysaccharides from Streptococcus pneumoniae serotype 16F, and polysaccharide protein conjugates having this serotype.
  • the present invention is based, in part, on the structural identification of capsular polysaccharides from this serotype.
  • the present invention provides a polysaccharide with the following repeating unit: glycerol- I-PO 4
  • glycerol- I-PO 4 OAc(x) where x indicates a molar ratio of 0.0 to 1.0.
  • a polysaccharide from Streptococcus pneumoniae serotype 16F can be represented by
  • n the number of repeating units.
  • the polysaccharide has between 10 and 5,000 repeating units. In certain aspects, the polysaccharide has between 50 and 4,500, 150 and 4,500, or 150 to
  • the polysaccharide has a molecular weight from 50 kDa to 4,000 kDa. In certain aspects, the polysaccharide has a molecular weight from 100 kDa to
  • the S. pneumoniae serotype 16F polysaccharide has a molar ratio of O-acetyl groups to serotype 16F repeating unit of 0.0 to 1.0, e.g., at least any of
  • the present invention further provides activated polysaccharides produced from any of the above embodiments wherein the polysaccharide is activated with a chemical reagent to produce reactive groups for conjugation to a linker or carrier protein.
  • the activation occurs on one or more of the glycerol-l-P0 4 sugars.
  • the polysaccharide is activated with periodate.
  • the activation occurs on the carbon 2 position of either or both of the glycerol-1-
  • the present invention further provides polysaccharide-protein conjugates in which polysaccharides or activated polysaccharides as provided for above are conjugated to a carrier protein.
  • the carrier protein is selected from CRM197, diphtheria toxin fragment B (DTFB), DTFB C8, Diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa.
  • the carrier protein is CRM197.
  • the polysaccharide-protein conjugates are prepared using reductive animation chemistry under aqueous conditions or in an aprotic solvent such as dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • the polysaccharide-protein conjugates are prepared using reductive animation chemistry in DMSO.
  • the present invention provides a multivalent immunogenic composition
  • a multivalent immunogenic composition comprising unconjugated polysaccharides or polysaccharide-protein conjugates from Streptococcus pneumoniae serotypes 16F and unconjugated polysaccharides or polysaccharide-protein conjugates from one or more of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 17F, 18B, 18C, 19A, 19F, 20, 21, 22A, 22F, 23 A, 23B, 23F, 24F, 24B, 27, 28A, 31, 33F, 34, 35A, 35B, 35F, and 38.
  • a multivalent immunogenic composition comprises unconjugated polysaccharides or polysaccharide-carrier protein conjugates but not both. In one subembodiment, a multivalent immunogenic composition comprises a mixture of unconjugated polysaccharides or polysaccharide-carrier protein conjugates. In certain subembodiments, a multivalent immunogenic composition of the invention has up to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 serotypes.
  • Figure 1 depicts graphical representations of the repeating unit structure of S. pneumoniae serotype 16F polysaccharide.
  • the activation sites for periodate are shown in the ovals.
  • For an activated polysaccharide not all of the repeating unit's activation sites are activated. This reflects the carbon 2 position of the glycerol- I-PO 4 sugars.
  • Figures 2A-B depict A: the 600 MHz one-dimensional X H NMR spectrum of the capsular polysaccharide from S. pneumoniae serotype 16F in deuterium oxide (D 2 0) at 50°C. Signals arising from internal standards (DMSO and DSS-de), residual water (HOD), ethanol (EtOH), isopropanol (IP A) and acetate are marked. Minor signals marked by * are due to S. pneumoniae cell wall residuals such as C-polysaccharide and/or peptidoglycans.
  • B the 600 MHz one- dimensional X H NMR spectrum of oxidized and derivatized capsular polysaccharide from S.
  • the inset is an expansion of the imine signal formed by derivatization with thiosemicarbazide. Signals arising from internal standards (DMSO and DSS-d6) residual water (HOD) and buffer (Citrate) are marked.
  • Figure 3 depicts the one-dimensional (ID) X H NMR identity region to be used for serotype identifications of S. pneumoniae serotype 16F. Signal positions of each anomeric proton of the repeating unit from each monosaccharide residue is marked.
  • Figure 4 depicts partial two-dimensional (2D) X H - 1 C multiple bond correlation NMR spectrum of de-O-acetylated S. pneumoniae serotype 16F establishing covalent linkages between sugar residues in the repeating structure. Correlation establishing glycosidic linkages are labeled in the figure.
  • Figure 5 depicts establishment of phosphodiester linkages in the capsular polysaccharide repeating unit of S. pneumoniae serotype 16F.
  • Figure 6 depicts partial ID (top) and 2D X H - 1 C correlation NMR spectrum of oxidized and derivatized S. pneumoniae serotype 16F.
  • Figure 7 depicts partial ID (top) and 2D X H - 1 C correlation NMR spectrum of oxidized and derivatized S. pneumoniae serotype 16F showing only signals from methylene carbons. The additional methylene signals from the derivatization are circled.
  • Figures 8A-8E depict composite of NMR data acquired at 600 MHz of oxidized purified serotype 16F polysaccharide and derivatized with thiosemicarbazide.
  • D and E 2D X H - 1 C correlation spectrum showing the carbon chemical shift of the cross peaks (D) and the imine (E).
  • Figure 9 shows ELISA IgG antibody titers (post-dose 2) for rabbits immunized with S.
  • Figure 10 shows serotype specific OPA titers (post-dose 2) for rabbits immunized with S.
  • FIG. 11 shows serotype specific (S. pneumoniae serotypes 16F, 23 A, 23B, 24F, 31) pre- immune, PD1 and PD2 geometric mean antibody titers for rabbits immunized with a multivalent pneumococcal conjugate vaccine (2 ⁇ g/PnPs). Error bars represent 2 standard errors of the geometric mean titer of each serotype (X-axis).
  • Figure 12 shows serotype specific (S. pneumoniae serotypes 16F, 23 A, 23B, 24F, 31) pre- immune, PD1 and PD2 OP A dilution titers for rabbits immunized with a multivalent
  • the present invention is based, in part, on the identification of novel pneumococcal polysaccharide structure(s) by NMR technology. It is believed that the structure provided herein is the first identification or the first correct identification of S. pneumoniae serotype 16F.
  • the S. pneumoniae serotype 16F polysaccharide was produced from its respective strain and purified. The produced (and purified) polysaccharide was used to generate polysaccharide-CRM197 conjugate. S. pneumoniae serotype 16F has a unique polysaccharide structure, which results in a unique conjugate production process. The resulting conjugate was demonstrated to be immunogenic in animal studies.
  • polysaccharide is meant to include any antigenic saccharide element (or antigenic unit) commonly used in the immunologic and bacterial vaccine arts, including, but not limited to, a “saccharide”, an “oligosaccharide”, a “polysaccharide”, a “liposaccharide”, a “lipo-oligosaccharide (LOS)", a “lipopolysaccharide (LPS)", a "glycosylate”, a “glycoconjugate”, a "derivatized or activated polysaccharide or oligosaccharide", and the like.
  • a saccharide element or antigenic unit
  • immunogenic composition refers to a composition containing an antigen, such as a bacterial capsular polysaccharide or a polysaccharide-protein conjugate, that has the ability to elicit an immune response in a host such as a mammal, either humorally or cellularly mediated, or both.
  • the immunogenic composition may serve to sensitize the host by the presentation of the antigen in association with MHC molecules at a cell surface.
  • antigen-specific T-cells or antibodies can be generated to allow for the future protection of an immunized host. Immunogenic compositions thus can protect the host from infection by the bacteria, reduced severity, or may protect the host from death due to the bacterial infection. .
  • Immunogenic compositions may also be used to generate polyclonal or monoclonal antibodies, which may be used to confer passive immunity to a subject. Immunogenic compositions may also be used to generate antibodies that are functional as measured by the killing of bacteria in either an animal efficacy model or via an opsonophagocytic killing assay.
  • isolated in connection with a polysaccharide refers to isolation of S. pneumoniae serotype specific capsular polysaccharide from purified
  • an isolated polysaccharide refers to partial removal of proteins, nucleic acids and non-specific endogenous polysaccharide (C- poly saccharide).
  • the isolated polysaccharide contains less than 10%, 8%, 6%, 4%, or 2% protein impurities and/or nucleic acids.
  • the isolated polysaccharide contains less than 20% of C-polysaccharide with respect to type specific polysaccharides.
  • purified in connection with a bacterial capsular polysaccharide refers to the purification of the polysaccharide from cell lysate through means such as centrifugation, precipitation, and ultra-filtration.
  • a purified polysaccharide refers to removal of cell debris and DNA.
  • Mw refers to the weight averaged molecular weight and is typically expressed in Da or kDa. Mw takes into account that a bigger molecule contains more of the total mass of a polymer sample than the smaller molecules do. Mw can be determined by techniques such as static light scattering, small angle neutron scattering, X-ray scattering, and sedimentation velocity.
  • Mn refers to a number average molecular weight and is typically expressed in Da or kDa. Mn is calculated by taking the total weight of a sample divided by the number of molecules in the sample and can be determined by techniques such as gel permeation chromatography, viscometry via the (Mark-Houwink equation), colligative methods such as vapor pressure osmometry, end-group determination or proton NMR. Mw/Mn reflects polydispersity.
  • molar ratio is a fraction typically expressed as a decimal to the tenths or hundredths place.
  • a molar ratio of from 0 or 0.1 to 1.0 expressed in tenths will include any of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0.
  • PnPs refers to pneumococcal polysaccharide.
  • the term “comprises” when used with the immunogenic composition of the invention refers to the inclusion of any other components (subject to limitations of “consisting of language for the antigen mixture), such as adjuvants and excipients.
  • the term “consisting of when used with the multivalent polysaccharide-protein conjugate mixture of the invention refers to a mixture having those particular S. pneumoniae polysaccharide protein conjugates and no other S. pneumoniae polysaccharide protein conjugates from a different serotype.
  • activation site on a sugar means that the site can be chemically modified to form a reactive group. Activation site takes into account the preferred tendency of an activation agent to react at a specific site.
  • activated polysaccharide refers to a polysaccharide that has been chemically modified to form reactive groups in a polysaccharide chain.
  • An activated polysccharide does not necessarily mean that all the available activation sites have been chemically modified.
  • the phrase "extent of activation" on a polysaccharide chain refers to the overall ratio between the number of activated chemical group to the number of repeat units on the polysaccharide chain.
  • Gal galactose
  • Rha rhamnose
  • GlcN glucosamine
  • GalN galactosamine
  • Gro-P glycerol phosphate
  • the S. pneumoniae 16F structure shows the presence of an O-acetyl group on the ⁇ -Rha.
  • the analysis in the Examples shows that the O-Ac group is present on approximately 90% of the repeating units on the polysaccharide.
  • the 16F serotype polysaccharide has 0 (i.e., completely de-O-acetylated), or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9 mM acetate per mM of serotype 16F polysaccharide.
  • the identification of the structure for this serotype may allow its incorporation into pneumococcal vaccines, either unconjugated or as a polysaccharide-protein conjugate.
  • Conjugate vaccines comprising streptococcal and pneumococcal polysaccharides are well-known in the art. See e.g., U.S. Pat. Nos. 6,248,570; 5,866,135; and 5,773,007.
  • Capsular polysaccharides from Steptococcus pneumoniae from the serotype of the invention can be prepared by standard techniques known to those skilled in the art.
  • polysaccharides can be isolated from bacteria and may be sized to some degree by known methods (see, e.g., European Patent Nos. EP497524 and EP497525); and preferably by microfluidisation accomplished using a homogenizer or by chemical hydrolysis.
  • S. pneumoniae strains corresponding to each polysaccharide serotype are grown in a soy-based medium. The individual polysaccharides are then purified through standard steps including centrifugation, precipitation, and ultra-filtration. See, e.g., U.S. Patent Application Publication No.
  • Polysaccharides can be sized in order to reduce viscosity and/or to improve filterability of subsequent conjugated products.
  • Chemical hydrolysis may be conducted using acetic acid.
  • Mechanical sizing may be conducted using High Pressure Homogenization Shearing.
  • the purified polysaccharides before conjugation have a molecular weight of between 5 kDa and 4,000 kDa.
  • Molecular weight can be calculated by size exclusion chromatography (SEC) combined with multiangle light scattering detector (MALS) and refractive index detector (RI).
  • the polysaccharide has an average molecular weight of between 10 kDa and 4,000 kDa; between 50 kDa and 4,000 kDa; between 50 kDa and 3,000 kDa; between 50 kDa and 2,000 kDa; between 50 kDa and 1,500 kDa; between 50 kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDa and 500 kDa; between 80 kDa and 2000 kDa; between 100 kDa and 4,000 kDa; between 100 kDa and 3,000 kDa; 100 kDa and 2,000 kDa; between 100 kDa and 1,500 kDa; between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100 kDa and 500 kDa; between 100 and 400 kDa; between 100 kDa and 300
  • the S. pneumoniae serotype 16F polysaccharide has between 10 and 5000 repeating units. In certain aspects, the polysaccharide has between 50 and 3000, 100 to 2500, or 100 to 2000. In certain embodiments, the polysaccharide from serotype 16F has between 100 and 300 repeating units or between 128 and 256 repeating units.
  • the S. pneumoniae serotype 16F polysaccharide has a molar ratio of O-acetyl groups to serotype 16F repeating unit of 0.0-1.0, e.g., at least any of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 up to 1.0.
  • Polysaccharides from one or more of the S. pneumoniae serotypes can be conjugated to a carrier protein ("Pr") to improve immunogenicity in children, the elderly and/or immunocompromised subjects.
  • the serotypes may be prepared with the same carrier protein or different carrier proteins.
  • Each capsular polysaccharide of the same serotype is typically conjugated to the same carrier protein.
  • CRM197 is used as a carrier protein.
  • CRM 197 is a non-toxic variant of diphtheria toxin (DT).
  • the CRM 197 carrier protein is a mutant form of DT that is rendered non-toxic by a single amino acid substitution in Fragment A at residue 52.
  • the CRM197 carrier protein is isolated from cultures of Corynebacterium diphtheria strain C7 ( ⁇ 197) grown in casamino acids and yeast extract-based medium.
  • CRM197 is prepared recombinantly in accordance with the methods described in U.S. Pat. No. 5,614,382.
  • CRM197 is purified through a combination of ultra-filtration, ammonium sulfate precipitation, and ion- exchange chromatography.
  • CRM197 is prepared in Pseudomonas fluorescens using Pfenex Expression TechnologyTM (Pfenex Inc., San Diego, CA).
  • Suitable carrier proteins include additional inactivated bacterial toxins such as DT, Diphtheria toxoid fragment B (DTFB), TT (tetanus toxid) or fragment C of TT, pertussis toxoid, cholera toxoid (e.g., as described in International Patent Application Publication No. WO 2004/083251), E. coli LT (heat-labile enterotoxin), E. coli ST (heat-stable enterotoxin), and exotoxin A from Pseudomonas aeruginosa.
  • DTFB Diphtheria toxoid fragment B
  • TT tetanus toxid
  • fragment C of TT fragment C of TT
  • pertussis toxoid e.g., as described in International Patent Application Publication No. WO 2004/083251
  • E. coli LT heat-labile enterotoxin
  • E. coli ST heat
  • Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumococcal surface protein A (PspA; See International Application Patent Publication No. WO 02/091998), pneumococcal adhesin protein (PsaA), C5a peptidase from Group A or Group B streptococcus, or Haemophilus influenzae protein D, pneumococcal pneumolysin (Kuo et al., 1995, Infect Immun 63; 2706-13) including ply detoxified in some fashion for example dPLY-GMBS (See International Patent Application Publication No.
  • OMPC outer membrane complex c
  • porins porins
  • transferrin binding proteins pneumococcal surface protein A
  • PspA pneumococcal adhesin protein
  • C5a peptidase from Group A or Group B streptococcus or Haemophilus influenzae protein D
  • EP0471177 cytokines, lymphokines, growth factors or hormones
  • artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen derived antigens (See Falugi et al., 2001, Eur J Immunol 31 :3816-3824) such as N19 protein (See Baraldoi et al, 2004, Infect Immun 72:4884-7), iron uptake proteins (See International Patent Application Publication No. WO 01/72337), toxin A or B of C. difficile (See International Patent Publication No. WO 00/61761), and flagellin (See Ben- Yedidia et al., 1998, Immunol Lett 64:9) can also be used as carrier proteins.
  • DT mutants can also be used as the carrier protein, such as CRM176, CRM228, CRM45 (Uchida et al. , 1973, J Biol Chem 218:3838-3844); CRM9, CRM45, CRM 102, CRM 103 and CRM 107 and other mutations described by Nicholls and Youle in
  • a second carrier protein can be used for one or more of the antigens.
  • the second carrier protein is preferably a protein that is non-toxic and non-reactogenic and obtainable in sufficient amount and purity.
  • the second carrier protein is also conjugated or joined with an antigen, e.g., a S. pneumoniae polysaccharide to enhance immunogenicity of the antigen.
  • Carrier proteins should be amenable to standard conjugation procedures.
  • each capsular polysaccharide not conjugated to the first carrier protein is conjugated to the same second carrier protein (e.g., each capsular polysaccharide molecule being conjugated to a single carrier protein).
  • capsular polysaccharides not conjugated to the first carrier protein are conjugated to two or more carrier proteins (each capsular polysaccharide molecule being conjugated to a single carrier protein).
  • each capsular polysaccharide of the same serotype is typically conjugated to the same carrier protein.
  • the purified polysaccharides Prior to conjugation, can be chemically activated to make the saccharides capable of reacting with the carrier protein to form an activated polysaccharide.
  • activated polysaccharide refers to a polysaccharide that has been chemically modified as described below to enable conjugation to a linker or a carrier protein.
  • the purified polysaccharides can optionally be connected to a linker. Once activated or connected to a linker, each capsular polysaccharide is separately conjugated to a carrier protein to form a glycoconjugate.
  • the polysaccharide conjugates may be prepared by known coupling techniques.
  • the activation of S. pneumoniae serotypel6F polysaccharide occurs on one or more of the glycerol- I-PO4 sugars. In certain embodiments, the polysaccharide is activated with periodate. In certain aspects of this embodiment, the activation occurs on the carbon 2 position of either or both of the glycerol- I-PO4 sugars.
  • the polysaccharide can be coupled to a linker to form a polysaccharide-linker intermediate in which the free terminus of the linker is an ester group.
  • the linker is therefore one in which at least one terminus is an ester group. The other terminus is selected so that it can react with the polysaccharide to form the polysaccharide-linker intermediate.
  • the coupling can also take place indirectly, i.e. with an additional linker that is used to derivatise the polysaccharide prior to coupling to the linker.
  • the polysaccharide is coupled to the additional linker using a carbonyl group at the reducing terminus of the polysaccharide.
  • This coupling comprises two steps: (al) reacting the carbonyl group with the additional linker; and (a2) reacting the free terminus of the additional linker with the linker.
  • the additional linker typically has a primary amine group at both termini, thereby allowing step (al) to take place by reacting one of the primary amine groups with the carbonyl group in the polysaccharide by reductive amination.
  • a primary amine group is used that is reactive with the carbonyl group in the polysaccharide. Hydrazide or hydroxylamino groups are suitable. The same primary amine group is typically present at both termini of the additional linker. The reaction results in a polysaccharide-additional linker intermediate in which the polysaccharide is coupled to the additional linker via a C— N linkage.
  • the polysaccharide can be coupled to the additional linker using a different group in the polysaccharide, particularly a carboxyl group.
  • This coupling comprises two steps: (al) reacting the group with the additional linker; and (a2) reacting the free terminus of the additional linker with the linker.
  • the additional linker typically has a primary amine group at both termini, thereby allowing step (al) to take place by reacting one of the primary amine groups with the carboxyl group in the polysaccharide by ED AC activation.
  • a primary amine group is used that is reactive with the EDAC-activated carboxyl group in the polysaccharide.
  • a hydrazide group is suitable.
  • the same primary amine group is typically present at both termini of the additional linker.
  • the reaction results in a polysaccharide- additional linker intermediate in which the polysaccharide is coupled to the additional linker via an amide linkage.
  • the chemical activation of the polysaccharides and subsequent conjugation to the carrier protein by reductive amination can be achieved by means described in U.S. Pat. Nos. 4,365,170, 4,673,574 and 4,902,506, U.S. Patent Application Publication Nos. 2006/0228380, 2007/184072, 2007/0231340 and 2007/0184071, and
  • the chemistry may entail the activation of pneumococcal polysaccharide by reaction with any oxidizing agent which a primary hydroxyl group to an aldehyde, such as
  • TEMPO in the presence of oxidant (W02104/097099), or reacting two vicinal hydroxyl groups to aldehydes, such as periodate (including sodium periodate, potassium periodate, or periodic acid).
  • aldehydes such as periodate (including sodium periodate, potassium periodate, or periodic acid).
  • the reactions lead to an oxidation of primary hydroxyl groups or oxidative cleavage of vicinal hydroxyl groups of the carbohydrates with the formation of reactive aldehyde groups.
  • coupling to the carrier protein is by reductive amination via direct amination to the lysyl groups of the protein.
  • conjugation is carried out by reacting a mixture of the activated polysaccharide and carrier protein with a reducing agent such as sodium cyanoborohydride in the presence of nickel.
  • the conjugation reaction may take place under aqueous solution or in the presence of dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • Unreacted aldehydes are then capped with the addition of a strong reducing agent, such as sodium borohydride.
  • Reductive amination involves two steps, (1 ) oxidation of the polysaccharide to form reactive aldehydes, (2) reduction of the imine (Schiff base) formed between activated polysaccharide and a carrier protein to form a stable amine conjugate bond.
  • the polysaccharide is optionally size reduced. Mechanical methods (e.g. homogenization) or chemical hydrolysis may be employed. Chemical hydrolysis maybe conducted using acetic acid.
  • the oxidation step may involve reaction with periodate.
  • periodate includes both periodate and periodic acid; the term also includes both metaperiodate (I(V) and orthoperiodate ( ⁇ 5 ) and includes the various salts of periodate (e.g. , sodium periodate and potassium periodate).
  • polysaccharide is oxidized in the presence of metaperiodate, preferably in the presence of sodium periodate (NalC ⁇ ).
  • capsular polysaccharide is oxydized in the presence of orthoperiodate, preferably in the presence of periodic acid.
  • the oxidizing agent is a stable nitroxyl or nitroxide radical compound, such as piperidine-N-oxy or pyrrolidine-N-oxy compounds, in the presence of an oxidant to selectively oxidize primary hydroxyls (as described in, for example, International
  • the actual oxidant is the N-oxoammonium salt, in a catalytic cycle.
  • said stable nitroxyl or nitroxide radical compound are piperidine-N-oxy or pyrrolidine-N-oxy compounds.
  • said stable nitroxyl or nitroxide radical compound bears a TEMPO (2,2,6,6-tetramethyl-l-piperidinyloxy) or a PROXYL (2,2,5, 5-tetramethyl-l -pyrrolidinyloxy) moiety.
  • said stable nitroxyl radical compound is TEMPO or a derivative thereof.
  • said oxidant is a molecule bearing a N-halo moiety.
  • said oxidant is selected from the group consisting of N- ChloroSuccinimide, N-Bromosuccinimide, N-Iodosuccinimide, Dichloroisocyanuric acid, 1,3,5- trichloro-1 ,3,5-triazinane-2,4,6-trione, Dibromoisocyanuric acid, 1,3,5-tribromo-l ,3,5- triazinane-2,4,6-trione, Diiodoisocyanuric acid and 1 ,3,5-triiodo-l,3,5-triazinane-2,4,6-trione.
  • said oxidant is N- Chlorosuccinimide.
  • the oxidizing agent is 2,2,6,6-Tetramethyl-l -piperidinyloxy (TEMPO) free radical and N-Chlorosuccinimide (NCS) as the cooxidant (as described in International Patent Application Publication No. WO2014/097099). Therefore in one aspect, the glycoconjugates from S.
  • TEMPO 2,2,6,6-Tetramethyl-l -piperidinyloxy
  • NCS N-Chlorosuccinimide
  • pneumoniae are obtainable by a method comprising the steps of: a) reacting a saccharide with 2,2,6,6-tetramethyl-l -piperidinyloxy (TEMPO) and N- chlorosuccinimide (NCS) in an aqueous solvent to produce an activated saccharide; and b) reacting the activated saccharide with a carrier protein comprising one or more amine groups (said method is designated "TEMPO/NCS-reductive animation" thereafter).
  • TEMPO 2,2,6,6-tetramethyl-l -piperidinyloxy
  • NCS N- chlorosuccinimide
  • the oxidation reaction is quenched by addition of a quenching agent.
  • the quenching agent maybe selected from vicinal diols, 1 ,2-aminoalcohols, amino acids, glutathione, sulfite, bisulfate, dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites or phosphorous acid (such as glycerol, ethylene glycol, propan-1 ,2-diol, butan-1 ,2-diol or butan- 2,3-diol, ascorbic acid).
  • the second step of the conjugation process for reductive animation is the reduction of the imine (Schiff base) bond between activated polysaccharide and a carrier protein to form a stable conjugate bond (so-called reductive amination), using a reducing agent.
  • Reducing agents which are suitable include the cyanoborohydrides (such as sodium
  • cyanoborohydride or sodium borohydride.
  • the reducing agent is sodium cyanoborohydride.
  • the reductive amination reaction is carried out in aprotic solvent (or a mixture of aprotic solvents).
  • the reduction reaction is carried out in DMSO (dimethyl sulfoxide) or in DMF
  • DMSO dimethylformamide
  • DMF solvent may be used to reconstitute the activated polysaccharide and carrier protein, if lyophilized.
  • the aprotic solvent is DMSO.
  • this capping or quenching agent is sodium borohydride (NaBH 4 ).
  • Suitable alternatives include sodium triacetoxyborohydride or sodium or zinc borohydride in the presence of Bronsted or Lewis acids), amine boranes such as pyridine borane, 2-Picoline Borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe'PrN-BL , benzylamine-BH 3 or 5-ethyl-2-methylpyridine borane (PEMB) or borohydride exchange resin.
  • amine boranes such as pyridine borane, 2-Picoline Borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe'PrN-BL , benzylamine-BH 3 or 5-ethyl-2-methylpyridine borane (PEMB) or borohydride exchange resin.
  • Glycoconjugates prepared using reductive amination in an aprotic solvent are generally used in multivalent pneumococcal conjugate vaccines.
  • the reduction reaction for the remaining seroytpes is carried out in aqueous solvent (e.g., selected from PBS (phosphate buffered saline), MES (2-(N-morpholino)ethanesulfonic acid), HEPES, (4- (2-hydroxy ethyl)- 1-piperazineethanesulfonic acid), Bis-tris, ADA (N-(2- Acetamido)iminodiacetic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), MOPSO (3-Morpholino-2-hydroxypropanesulfonic acid), BES (N,N-bis(2 -hydroxy ethyl)-2- aminoethan
  • aqueous solvent e.g., selected from PBS (phosphate buffered sa
  • the glycoconjugates of the present invention comprise a polysaccharide having a molecular weight of between 10 kDa and 10,000 kDa. In other such embodiments, the polysaccharide has a molecular weight of between 25 kDa and 5,000 kDa. In other such embodiments, the polysaccharide has a molecular weight of between 50 kDa and 1,000 kDa. In other such embodiments, the polysaccharide has a molecular weight of between 70 kDa and 900 kDa. In other such embodiments, the polysaccharide has a molecular weight of between 100 kDa and 800 kDa.
  • the polysaccharide has a molecular weight of between 200 kDa and 600 kDa. In further such embodiments, the polysaccharide has a molecular weight of 100 kDa to 1000 kDa; 100 kDa to 900 kDa; 100 kDa to 800 kDa; 100 kDa to 700 kDa; 100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa; 100 kDa to 300 kDa; 150 kDa to 1,000 kDa; 150 kDa to 900 kDa; 150 kDa to 800 kDa; 150 kDa to 700 kDa; 150 kDa to 600 kDa; 150 kDa to 500 kDa; 150 kDa to 400 kDa; 150 kDa to 300 kDa; 200 kDa to 1,000 kDa; 150
  • the conjugation reaction is performed by reductive animation wherein nickel is used for greater conjugation reaction efficiency and to aid in free cyanide removal.
  • Transition metals are known to form stable complexes with cyanide and are known to improve reductive methylation of protein amino groups and formaldehyde with sodium cyanoborohydride (S Gidley et al., Biochem J. 1982, 203: 331-334; Jentoft et al. Anal Biochem. 1980, 106: 186-190).
  • the addition of nickel increases the consumption of protein during the conjugation of and leads to formation of larger, potentially more immieuxic conjugates.
  • Suitable alternative chemistries include the activation of the saccharide with 1- cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester.
  • the activated saccharide may thus be coupled directly or via a spacer (linker) group to an amino group on the carrier protein.
  • the spacer could be cystamine or cysteamine to give a thiolated polysaccharide which could be coupled to the carrier via a thioether linkage obtained after reaction with a maleimide-activated carrier protein (for example using GMBS) or a haloacetylated carrier protein (for example using iodoacetimide [e.g.
  • the cyanate ester (optionally made by CDAP chemistry) is coupled with hexane diamine or adipic acid dihydrazide (ADH) and the amino-derivatised saccharide is conjugated to the carrier protein using carbodiimide (e.g. ED AC or EDC) chemistry via a carboxyl group on the protein carrier.
  • carbodiimide e.g. ED AC or EDC
  • the glycoconjugates may be purified (enriched with respect to the amount of polysaccharide-protein conjugate) by a variety of techniques known to the skilled person. These techniques include dialysis, concentration/diafiltration operations, tangential flow filtration, ultrafiltration, precipitation/elution, column chromatography (ion exchange chromatography, multimodal ion exchange chromatography, DEAE, or hydrophobic interaction chromatography), and depth filtration. See, e.g., U.S. Pat. No. 6,146,902.
  • the glycoconjugates are purified by diafilitration or ion exchange chromatography or size exclusion chromatography.
  • One way to characterize the glycoconjugates of the invention is by the number of lysine residues in the carrier protein (e.g., CRM197) that become conjugated to the saccharide, which can be characterized as a range of conjugated lysines (degree of conjugation).
  • the evidence for lysine modification of the carrier protein, due to covalent linkages to the polysaccharides, can be obtained by amino acid analysis using routine methods known to those of skill in the art. Conjugation results in a reduction in the number of lysine residues recovered, compared to the carrier protein starting material used to generate the conjugate materials.
  • the degree of conjugation of the glycoconjugate of the invention is between 2 and 15, between 2 and 13, between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4, between 3 and 15, between 3 and 13, between 3 and 10, between 3 and 8, between 3 and 6, between 3 and 5, between 3 and 4, between 5 and 15, between 5 and 10, between 8 and 15, between 8 and 12, between 10 and 15 or between 10 and 12.
  • the degree of conjugation of the glycoconjugate of the invention is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 1, about 12, about 13, about 14 or about 15.
  • glycoconjugate of the invention is between 4 and 7.
  • the carrier protein is CRM197.
  • polysaccharide to carrier protein in the glycoconjugate is between 0.5 and 3.0 (e.g., about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1 , about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1 , about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.0).
  • the saccharide to carrier protein ratio is between 0.5 and 2.0, between 0.5 and 1.5, between 0.8 and 1.2, between 0.5 and 1.0, between 1.0 and 1.5 or between 1.0 and 2.0. In further embodiments, the saccharide to carrier protein ratio (w/w) is between 0.8 and 1.2. In a preferred embodiment, the ratio of capsular polysaccharide to carrier protein in the conjugate is between 0.9 and 1.1. In some such embodiments, the carrier protein is CRM197.
  • the glycoconjugates and immunogenic compositions of the invention may contain free saccharide that is not covalently conjugated to the carrier protein, but is nevertheless present in the glycoconjugate composition. The free saccharide may be non-covalently associated with (i.e., non-covalently bound to, adsorbed to, or entrapped in or with) the glycoconjugate.
  • the glycoconjugate comprises less than about 50%, 45%, 40%, 35%, 30%, 25%, 20% or 15% of free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment the glycoconjugate comprises less than about 25% of free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment the glycoconjugate comprises less than about 20% of free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment the glycoconjugate comprises less than about 15% of free polysaccharide compared to the total amount of polysaccharide.
  • multivalent polysaccharide vaccines comprise unconjugated S. pneumoniae serotype 16F polysaccharide and/or polysaccharide- protein conjugates from serotype 16F and capsular polysaccharides from one or more of S.
  • the immunogenic composition comprises, consists essentially of, or consists of capsular polysaccharides from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 S. pneumoniae serotypes individually conjugated to one or more carrier proteins.
  • saccharides from a particular serotype are not conjugated to more than one carrier protein.
  • compositions including pharmaceutical, immunogenic and vaccine compositions, comprising, consisting essentially of, or alternatively, consisting of any of the polysaccharide S. pneumoniae serotype combinations described above together with a pharmaceutically acceptable carrier and an adjuvant.
  • Formulation of the polysacchari de-protein conjugates of the present invention can be accomplished using art-recognized methods.
  • individual pneumococcal conjugates can be formulated with a physiologically acceptable vehicle to prepare the composition.
  • physiologically acceptable vehicles include, but are not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.
  • the vaccine composition is formulated in L-histidine buffer with sodium chloride.
  • an "adjuvant” is a substance that serves to enhance the immunogenicity of an immunogenic composition of the invention.
  • An immune adjuvant may enhance an immune response to an antigen that is weakly immunogenic when administered alone, e.g., inducing no or weak antibody titers or cell-mediated immune response, increase antibody titers to the antigen, and/or lowers the dose of the antigen effective to achieve an immune response in the individual.
  • adjuvants are often given to boost the immune response and are well known to the skilled artisan.
  • Suitable adjuvants to enhance effectiveness of the composition include, but are not limited to:
  • aluminum salts such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.
  • oil-in-water emulsion formulations with or without other specific immunostimulating agents such as muramyl peptides (defined below) or bacterial cell wall components), such as, for example, (a) MF59 (International Patent Application Publication No.
  • WO 90/14837 containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE) formulated into submicron particles using a microfluidizer such as Model HOY microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10% Squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, (c) RibiTM adjuvant system (RAS), (Corixa, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of 3- O-deacylated monophosphorylipid A (MPLTM) described in U.S.
  • MPLTM 3- O-deacylated monophosphorylipid A
  • AGP is 2-[(R)-3- tetradecanoyloxytetradecanoylaminojethyl 2-Deoxy-4-0-phosphono-3-0-[(R)-3- tetradecanoyloxytetradecanoyl]-2-[(R)-3 ⁇ tetradecanoyloxytetradecanoylamino]-b-D- glucopyranoside, which is also known as 529 (formerly known as RC529), which is formulated as an aqueous form or as a stable emulsion;
  • cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12,
  • interferons e.g., gamma interferon
  • GM-CSF granulocyte macrophage colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • TNF tumor necrosis factor
  • complement such as a trimer of complement component C3d.
  • the adjuvant is a mixture of 2, 3, or more of the above adjuvants, e.g., SBAS2 (an oil-in-water emulsion also containing 3-deacylated monophosphoryl lipid A and QS21).
  • SBAS2 an oil-in-water emulsion also containing 3-deacylated monophosphoryl lipid A and QS21.
  • Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl- D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2-(l ',2'-dipalmitoyl-sn-glycero-3- hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.
  • thr-MDP N-acetyl-muramyl-L-threonyl- D-isoglutamine
  • MTP-PE N-acetyl-normuramyl-L-alanine-2-(l ',2'-dipalmitoyl-sn-glycero-3- hydroxyphosphoryloxy)-ethylamine
  • the adjuvant is an aluminum salt.
  • the aluminum salt adjuvant may be an alum-precipitated vaccine or an alum-adsorbed vaccine.
  • Aluminum-salt adjuvants are well known in the art and are described, for example, in Harlow, E. and D. Lane (1988; Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory) and Nicklas, W. (1992; Aluminum salts. Research in Immunology 143:489-493).
  • the aluminum salt includes, but is not limited to, hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, Alhydrogel ® , Superfos, Amphogel ® , aluminum (III) hydroxide, aluminum hydroxyphosphate (Aluminum Phosphate Adjuvant (APA)), amorphous alumina, trihydrated alumina, or trihydroxy aluminum.
  • APA is an aqueous suspension of aluminum hydroxyphosphate.
  • APA is manufactured by blending aluminum chloride and sodium phosphate in a 1 : 1 volumetric ratio to precipitate aluminum hydroxyphosphate. After the blending process, the material is size- reduced with a high-shear mixer to achieve a monodisperse particle size distribution. The product is then diafiltered against physiological saline and steam sterilized.
  • a commercially available Al(OH) 3 (e.g. Alhydrogel ® or Superfos of Denmark/ Accurate Chemical and Scientific Co., Westbury, NY) is used to adsorb proteins. Adsorption of protein is dependent, in another embodiment, on the pi (Isoelectric pH) of the protein and the pH of the medium. A protein with a lower pi adsorbs to the positively charged aluminum ion more strongly than a protein with a higher pi.
  • Aluminum salts may establish a depot of Ag that is released slowly over a period of 2-3 weeks, be involved in nonspecific activation of macrophages and complement activation, and/or stimulate innate immune mechanism (possibly through stimulation of uric acid). See, e.g., Lambrecht et al, 2009, Curr Opin Immunol 21 :23.
  • Monovalent bulk aqueous conjugates are typically blended together and diluted. Once diluted, the batch is sterile filtered.
  • Aluminum phosphate adjuvant is added aseptically to target a final concentration of 4 ⁇ g/mL for all S. pneumoniae serotypes except serotype 6B, which is diluted to a target of 8 ⁇ g/mL, and a final aluminum concentration of 250 ⁇ g/mL.
  • the adjuvanted, formulated batch will be filled into vials or syringes.
  • the adjuvant is a CpG-containing nucleotide sequence, for example, a CpG-containing oligonucleotide, in particular, a CpG-containing
  • the adjuvant is ODN 1826, which may be acquired from Coley Pharmaceutical Group.
  • CpG-containing nucleotide refers to a nucleotide molecule of 6-50 nucleotides in length that contains an unmethylated CpG moiety. See, e.g., Wang et al, 2003, Vaccine 21 :4297. In another embodiment, any other art-accepted definition of the terms is intended.
  • CpG-containing oligonucleotides include modified oligonucleotides using any synthetic intemucleoside linkages, modified base and/or modified sugar.
  • compositions and formulations of the present invention can be used to protect or treat a human susceptible to infection, e.g., a pneumococcal infection, by means of administering the vaccine via a systemic or mucosal route.
  • the present invention provides a method of inducing an immune response to a S. pneumoniae capsular polysaccharide conjugate, comprising administering to a human an immunologically effective amount of an immunogenic composition of the present invention.
  • the present invention provides a method of vaccinating a human against a pneumococcal infection, comprising the step of administering to the human an immunogically effective amount of an immunogenic composition of the present invention.
  • Optimal amounts of components for a particular vaccine can be ascertained by standard studies involving observation of appropriate immune responses in subjects.
  • the dosage for human vaccination is determined by extrapolation from animal studies to human data.
  • the dosage is determined empirically.
  • Effective amount of a composition of the invention refers to a dose required to elicit antibodies that significantly reduce the likelihood or severity of infectivitiy of a microbe, e.g., S. pneumoniae, during a subsequent challenge.
  • the methods of the invention can be used for the prevention and/or reduction of primary clinical syndromes caused by microbes, e.g., S. pneumoniae, including both invasive infections (meningitis, pneumonia, and bacteremia), and noninvasive infections (acute otitis media, and sinusitis).
  • microbes e.g., S. pneumoniae
  • invasive infections meningitis, pneumonia, and bacteremia
  • noninvasive infections acute otitis media, and sinusitis
  • compositions of the invention can include one or more of: injection via the intramuscular, intraperitoneal, intradermal or subcutaneous routes; or via mucosal administration to the oral/alimentary, respiratory or genitourinary tracts.
  • intranasal administration is used for the treatment of pneumonia or otitis media (as nasopharyngeal carriage of pneumococci can be more effectively prevented, thus attenuating infection at its earliest stage).
  • the amount of conjugate in each vaccine dose is selected as an amount that induces an immunoprotective response without significant, adverse effects. Such amount can vary depending upon the pneumococcal serotype.
  • each dose will comprise 0.1 to 100 ⁇ g of each polysaccharide, particularly 0.1 to 10 ⁇ g, and more particularly 1 to 5 ⁇ g.
  • each dose can comprise 100, 150, 200, 250, 300, 400, 500, or 750 ng or l, 1.5, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 25, 30, 40, 50, 60, 70, 80, 90, or 100 ⁇ g of each polysaccharide.
  • Optimal amounts of components for a particular vaccine can be ascertained by standard studies involving observation of appropriate immune responses in subjects.
  • the dosage for human vaccination is determined by extrapolation from animal studies to human data.
  • the dosage is determined empirically.
  • the dose of the aluminum salt is 10, 15, 20, 25, 30, 50, 70, 100, 125, 150, 200, 300, 500, or 700 ⁇ g, or 1, 1.2, 1.5, 2, 3, 5 mg or more.
  • the dose of aluminum salt described above is per ⁇ g of recombinant protein.
  • each 0.5 mL dose is formulated to contain: 2 ⁇ g of each S. pneumoniae polysaccharide, except for serotype 6B polysaccharide of 4 ⁇ g; about 32 ⁇ g CRM197 carrier protein (e.g., 32 ⁇ g ⁇ 5 ⁇ g, ⁇ 3 ⁇ g, ⁇ 2 ⁇ g, or ⁇ 1 ⁇ g); 0.125 mg of elemental aluminum (0.5 mg aluminum phosphate) adjuvant; and sodium chloride and L-histidine buffer.
  • the sodium chloride concentration is about 150 mM (e.g., 150 mM ⁇ 25 mM, ⁇ 20 mM, ⁇ 15 mM, ⁇ 10 mM, or ⁇ 5 mM) and about 20 mM (e..g, 20 mM ⁇ 5 mM, ⁇ 2.5 mM, ⁇ 2 mM, ⁇ 1 mM, or ⁇ 0.5 mM) L-histidine buffer.
  • the subject is human.
  • the human patient is an infant (less than 1 year of age), toddler (approximately 12 to 24 months), or young child (approximately 2 to 5 years).
  • the human patient is an elderly patient (> 65 years).
  • the compositions of this invention are also suitable for use with older children, adolescents and adults (e.g., aged 18 to 45 years or 18 to 65 years).
  • a composition of the present invention is administered as a single inoculation.
  • the composition is administered twice, three times or four times or more, adequately spaced apart.
  • the composition may be administered at 1, 2, 3, 4, 5, or 6 month intervals or any combination thereof.
  • the immunization schedule can follow that designated for pneumococcal vaccines.
  • the routine schedule for infants and toddlers against invasive disease caused by S. pneumoniae is 2, 4, 6 and 12-15 months of age.
  • the composition is administered as a 4-dose series at 2, 4, 6, and 12-15 months of age.
  • compositions of this invention may also include one or more proteins from S. pneumoniae.
  • S. pneumoniae proteins suitable for inclusion include those identified in International Patent Application Publication Nos. WO 02/083855 and WO 02/053761.
  • compositions of the invention can be administered to a subject by one or more method known to a person skilled in the art, such as parenterally, transmucosally, transdermally, intramuscularly, intravenously, intra-dermally, intra-nasally, subcutaneously, intra-peritonealy, and formulated accordingly.
  • compositions of the present invention are administered via epidermal injection, intramuscular injection, intravenous, intra-arterial, subcutaneous injection, or intra-respiratory mucosal injection of a liquid preparation.
  • Liquid formulations for injection include solutions and the like.
  • composition of the invention can be formulated as single dose vials, multi- dose vials or as pre-filled syringes.
  • compositions of the present invention are administered orally, and are thus formulated in a form suitable for oral administration, i.e., as a solid or a liquid preparation.
  • Solid oral formulations include tablets, capsules, pills, granules, pellets and the like.
  • Liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
  • compositions are aqueous or nonaqueous solutions, suspensions, emulsions or oils.
  • nonaqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.
  • the pharmaceutical composition may be isotonic, hypotonic or hypertonic.
  • a pharmaceutical composition for infusion or injection is essentially isotonic when it is administrated.
  • the pharmaceutical composition may preferably be isotonic or hypertonic. If the pharmaceutical composition is hypertonic for storage, it may be diluted to become an isotonic solution prior to administration.
  • the isotonic agent may be an ionic isotonic agent such as a salt or a non-ionic isotonic agent such as a carbohydrate.
  • ionic isotonic agents include but are not limited to NaCl, CaC ⁇ , KC1 and MgC ⁇ .
  • non-ionic isotonic agents include but are not limited to sucrose, trehalose, mannitol, sorbitol and glycerol.
  • At least one pharmaceutically acceptable additive is a buffer.
  • the composition comprises a buffer, which is capable of buffering a solution to a pH in the range of 4 to 10, such as 5 to 9, for example 6 to 8.
  • the buffer may for example be selected from the group consisting of Tris, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, L-histidine, glycine, succinate and triethanolamine buffer.
  • the buffer may furthermore for example be selected from USP compatible buffers for parenteral use, in particular, when the pharmaceutical formulation is for parenteral use.
  • the buffer may be selected from the group consisting of monobasic acids such as acetic, benzoic, gluconic, glyceric and lactic; dibasic acids such as aconitic, adipic, ascorbic, carbonic, glutamic, malic, succinic and tartaric, polybasic acids such as citric and phosphoric; and bases such as ammonia, diethanolamine, glycine, triethanolamine, and Tris.
  • Parenteral vehicles for subcutaneous, intravenous, intraarterial, or intramuscular inj ection
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like.
  • Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants.
  • water, saline, aqueous dextrose and related sugar solutions, glycols such as propylene glycols or polyethylene glycol, Polysorbate 80 (PS- 80), Polysorbate 20 (PS-20), and Poloxamer 188 (P I 88) are preferred liquid carriers, particularly for injectable solutions.
  • oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.
  • the formulations may also contain a surfactant.
  • Preferred surfactants include, but are not limited to: the polyoxy ethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially PS-20 and PS-80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAXTM tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-1,2- ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypoly ethoxy ethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40);
  • phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such as the TergitolTMNP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as the SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate.
  • a preferred surfactant for including in the emulsion is PS-20 or PS-80.
  • surfactants can be used, e.g. PS-80/Span 85 mixtures.
  • a combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (PS-80) and an octoxynol such as t-octylphenoxypoly ethoxy ethanol (Triton X-100) is also suitable.
  • Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol.
  • Preferred amounts of surfactants are: polyoxyethylene sorbitan esters (such as PS- 80) 0.01 to 1% w/v, in particular about 0.1% w/v; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) 0.001 to 0.1% w/v, in particular 0.005 to 0.02% w/v; polyoxyethylene ethers (such as laureth 9) 0.1 to 20% w/v, preferably 0.1 to 10% w/v and in particular 0.1 to 1% w/v or about 0.5% w/v.
  • polyoxyethylene sorbitan esters such as PS- 80
  • octyl- or nonylphenoxy polyoxyethanols such as Triton X-100, or other detergents in the Triton series
  • polyoxyethylene ethers such as laureth 9) 0.1 to 20% w/v, preferably 0.1 to 10% w/v and in
  • the composition consists essentially of L-histidine (20 mM), saline (150 mM) and 0.2% w/v PS-20 at a pH of 5.8 with 250 ⁇ g/mL of APA (Aluminum Phosphate Adjuvant).
  • PS-20 can range from 0.005 to 0.1% w/v with the presence of PS-20 or PS-80 in formulation controlling aggregation during simulated manufacture and in shipping using primary packaging.
  • Process consists of combining blend of up to 44 S. pneumoniae polysaccharide serotypes in L-histidine, sodium chloride, and PS-20 then combining this blended material with APA and sodium chloride with or without antimicrobial preservatives.
  • the choice of surfactant may need to be optimized for different drug products and drug substances.
  • PS-20 and P188 are preferred.
  • the choice of chemistry used to prepare the conjugate can also influence the stabilization of the formulation.
  • pneumococcal poly sacchari de-protein conjugates prepared in aqueous or DMSO solvent and combined in a multivalent composition show significant differences in stability depending on the particular surfactant systems used for formulation.
  • a poloxamer generally has a molecular weight in the range from 1,100 Da to 17,400 Da, from 7,500 Da to 15,000 Da, or from 7,500 Da to 10,000 Da.
  • the poloxamer can be selected from poloxamer 188 or poloxamer 407.
  • the final concentration of the poloxamer in the formulations of the invention is from 0.001 to 5% w/v, or 0.025 to 1% w/v.
  • a surfactant system comprising a poloxamer must further comprise a polyol.
  • the polyol is propylene glycol and is at final concentration from 1 to 20% w/v.
  • the polyol is polyethylene glycol 400 and is at final concentration from 1 to 20% w/v.
  • Suitable polyols for the formulations are polymeric polyols, particularly polyether diols including, but are not limited to, propylene glycol and polyethylene glycol, Polyethylene glycol monomethyl ethers.
  • Propylene glycol is available in a range of molecular weights of the monomer from -425 Da to -2,700 Da.
  • Polyethylene glycol and Polyethylene glycol monomethyl ether is also available in a range of molecular weights ranging from -200 Da to
  • a preferred polyethylene glycol is polyethylene glycol 400.
  • the final concentration of the polyol in the formulations may be 1 to 20% w/v or 6 to 20% w/v.
  • the formulation also contains a pH-buffered saline solution.
  • the buffer may, for example, be selected from the group consisting of Tris, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, L-histidine, glycine, succinate, HEPES (4-(2- hydroxyethyl)-l-piperazineethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), MES (2- ⁇ ⁇ 1 ⁇ )6 ⁇ ] ⁇ 3 ⁇ 68 ⁇ 1 ⁇ acid) and triethanolamine buffer.
  • the buffer is capable of buffering a solution to a pH in the range of 4 to 10, 5.2 to 7.5, or 5.8 to 7.0.
  • the buffer selected from the group consisting of phosphate, succinate, L-histidine, MES, MOPS, HEPES, acetate or citrate.
  • the buffer may furthermore, for example, be selected from USP compatible buffers for parenteral use, in particular, when the pharmaceutical formulation is for parenteral use.
  • the concentrations of buffer will range from 1 mM to 50 mM or 5 mM to 50 mM.
  • the buffer is L-histidine at a final concentration of 5 mM to 50 mM, or succinate at a final concentration of 1 mM to 10 mM.
  • the L-histidine is at a final concentration of 20 mM ⁇ 2 mM.
  • saline solution i.e., a solution containing NaCl
  • other salts suitable for formulation include but are not limited to, CaC ⁇ , KC1 and MgC ⁇ and combinations thereof.
  • Non-ionic isotonic agents including but not limited to sucrose, trehalose, mannitol, sorbitol and glycerol may be used in lieu of a salt.
  • Suitable salt ranges include, but not are limited to 25 mM to 500 mM or 40 mM to 170 mM.
  • the saline is NaCl, optionally present at a concentration from 20 mM to 170 mM.
  • the formulations comprise a L-histidine buffer with sodium chloride.
  • the pharmaceutical composition is delivered in a controlled release system.
  • the agent can be administered using intravenous infusion, a transdermal patch, liposomes, or other modes of administration.
  • polymeric materials are used; e.g. in microspheres in or an implant.
  • compositions of this invention may also include one or more proteins from S. pneumoniae.
  • S. pneumoniae proteins suitable for inclusion include those identified in International Patent Application Publication Nos. WO 02/083855 and WO 02/053761.
  • Conjugate samples are injected and separated by high performance size-exclusion chromatography (HPSEC). Detection is accomplished with ultraviolet (UV), multi-angle light scattering (MALS) and refractive index (RI) detectors in series. Protein concentration is calculated from UV280 using an extinction coefficient. Polysaccharide concentration is deconvoluted from the RI signal (contributed by both protein and polysaccharide) using the dn/dc factors which are the change in a solution's refractive index with a change in the solute concentration reported in mL/g. Average molecular weight of the samples are calculated by Astra software (Wyatt Technology Corporation, Santa Barbara, CA) using the measured concentration and light scattering information across the entire sample peak.
  • UV ultraviolet
  • MALS multi-angle light scattering
  • RI refractive index
  • molecular weight is the weight-average molecular weight. Determination of lysine consumption in conjugated protein as a measure of the number of covalent attachments between polysaccharide and carrier protein
  • the Waters AccQ-Tag amino acid analysis is used to measure the extent of conjugation in conjugate samples.
  • Samples are hydrolyzed using vapor phase acid hydrolysis in the Eldex workstation, to break the carrier proteins down into their component amino acids.
  • the free amino acids are derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC).
  • AQC 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate
  • the derivatized samples are then analyzed using UPLC with UV detection on a C18 column.
  • the average protein concentration is obtained using representative amino acids other than lysine.
  • Lysine consumption during conjugation i.e., lysine loss
  • Free polysaccharide i.e., polysaccharide that is not conjugated with CRM197
  • Free polysaccharide i.e., polysaccharide that is not conjugated with CRM197
  • DOC deoxycholate
  • hydrochloric acid Precipitates are then filtered out and the filtrates are analyzed for free polysaccharide concentration by HPSEC/UV/MALS/RI. Free polysaccharide is calculated as a percentage of total polysaccharide measured by HPSEC/UV/MALS/RI. Free protein testing
  • Free polysaccharide, polysaccharide-CRM197 conjugate, and free CRM197 in the conjugate samples are separated by capillary electrophoresis in micellar electrokinetic chromatography (MEKC) mode. Briefly, samples are mixed with MEKC running buffer containing 25 mM borate, 100 mM SDS, pH 9.3, and are separated in a preconditioned bare- fused silica capillary. Separation is monitored at 200 nm and free CRM197 is quantified with a CRM197 standard curve. Free protein results are reported as a percentage of total protein content determined by the HPSEC/UV/MALS/RI procedure.
  • pneumococcal capsular polysaccharides are also well known in the art. See, e.g., European Patent No. EP 0 497 524 Bl . The process described below generally follows the method described in European Patent No. EP 0 497 524 Bl and is generally applicable to all pneumococcal serotypes except where specifically modifed.
  • Isolates of pneumococcal subtype 16F were obtained from the Merck Culture Collection. Where needed, subtypes can be differentiated on the basis of Banling reaction using specific antisera. See, e.g., U.S. Pat. No. 5,847,112.
  • the obtained isolates were further clonally isolated by plating serially in two stages on agar plates consisting of an animal-component free medium containing soy peptone, yeast extract, and glucose without hemin.
  • Clonal isolates for each serotype were further expanded in liquid culture using animal-component free media containing soy peptone, yeast extract, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate, glucose, and glycerol to prepare the pre-master cell banks.
  • each serotype of pneumococcal polysaccharide consisted of a cell expansion and batch production fermentation followed by chemical inactivation prior to downstream purification.
  • a thawed cell bank vial from each serotype was expanded using a shake flask or culture bottle containing a pre-sterilized animal -component free growth media containing soy peptone or soy peptone ultrafiltrate, yeast extract or yeast extract ultrafiltrate, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate, and glucose.
  • the cell expansion culture was grown in a sealed shake flask or bottle to minimize gas exchange with temperature and agitation control.
  • a portion of the cell expansion culture was transferred to a production fermentor containing pre-sterilized animal-component free growth media containing soy peptone or soy peptone ultrafiltrate, yeast extract or yeast extract ultrafiltrate, sodium chloride, potassium phosphate, and glucose. Temperature, pH, pressure, and agitation were controlled. Airflow overlay was also controlled as sparging was not used.
  • the batch fermentation was terminated via the addition of a chemical inactivating agent, phenol, when glucose was nearly exhausted. Pure phenol was added to a final concentration of 0.8 - 1.2% to inactivate the cells and liberate the capsular polysaccharide from the cell wall.
  • the purification of the pneumococcal polysaccharide consisted of several centrifugation, depth filtration, concentration/diafiltration operations, and precipitation steps. All procedures were performed at room temperature unless otherwise specified.
  • Inactivated broth from the fermentor cultures of S. pneumoniae were flocculated with a cationic polymer (such as BPA-1000, Petrolite “Tretolite” and “Spectrum 8160” and poly(ethyleneimine), "Millipore pDADMAC”).
  • a cationic polymer such as BPA-1000, Petrolite “Tretolite” and “Spectrum 8160” and poly(ethyleneimine), "Millipore pDADMAC”
  • the cationic polymers binded to the impurity protein, nucleic acids and cell debris.
  • flocculated solids were removed via centrifugation and multiple depth filtration steps. Clarified broth was concentrated and diafiltered using a 100 kDa to 500 kDa MWCO (molecular weight cutoff) filter. Diafiltration was accomplished using Tris, MgC ⁇ buffer and sodium phosphate buffer. Diafiltration removed residual nucleic acid and protein.
  • the strategy for determining polysaccharide structure involved a multiple step process performed substantially as described in Abeygunawardana et al, Determination of the Chemical Structure of Complex Polysaccharides by Heteronuclear NMR Spectroscopy in Advances in Biophysical Chemistry 1993, Vol 3, pages 199-249, JAI Press Inc.
  • the purified polysaccharides were examined using standard ID and 2D NMR techniques.
  • polysaccharides from S. pneumoniae 16F serotype was identified as containing O-acetyl, a detailed analysis was performed on de-O-acetylated polysaccharide (O-acetate groups were removed using base hydrolysis). Finally, the polysaccharides were examined for the presence of phosphate using 1 P NMR.
  • TOCSY heteronuclear single quantum coherence spectroscopy
  • HSQC heteronuclear single quantum coherence spectroscopy
  • HSQC-TOCSY Multiplicity-edited HSQC was used to distinguish methylene from methine groups. Inter-residue linkages were determined through a combination of HMBC and NOESY spectroscopy.
  • the X H NMR spectrum of purified S. pneumoniae serotypel6F polysaccharide showed one O-methyl resonance indicating O-acetylation at one site.
  • the location of the O- acetyl group on the purified polysaccharide was determined by analyzing the chemical shift changes due to the effect of O-acetylation.
  • a large downfield X H chemical shift change of 0.8 - 0.5 ppm is indicative of O-acetyl substitution.
  • Long range C-H heteronuclear multiple bond correlation (HMBC) and 1 C NMR spectroscopy assigned the carbonyls to their respective residue.
  • ID Phosphorus NMR spectroscopy indicated serotype 16F polysaccharides contained phosphorus in the structure. Assignment of the phosphorus linkage sites were done through a combination of X H- 1 P HMBC spectroscopy and chemical shift differences to the de- phosphorylated form of the polysaccharide. Based on the NMR data in Figures 2A, 3, 4 and 5, the structure for the S.
  • pneumoniae capsular polysaccharide from serotype 16F was determined to be as follows: glycerol-l-P0 4
  • n represents the number of repeating units constituting the polysaccharide.
  • the sugar residues for the serotypel6F polysaccharide include rhamnose (Rha), glucose (Glc) and glycerol.
  • the italicized letters (p and f) refer to pyranose (a closed ring consisting of six atoms) and furanose (a closed ring consisting of five atoms). None of the sugars in the serotypel6F polysaccharide are in the form of furanose.
  • the a and ⁇ refer to the configuration of the proton attached to anomeric carbon of the sugar unit.
  • the anomeric carbon is always number 1 when labeling the carbon atom in a sugar unit (usually 1 through 6).
  • a means the anomeric proton is in the equatorial position in the 3D structure
  • ⁇ means the anomeric proton is in the axial position.
  • the numbers associated with arrows refer to how the individual sugar units are connected to each other.
  • a-Rha -(l ⁇ 3)-a-Glc/?- means the number 1 carbon of Rhamnose is linked to the number 3 carbon of Glucose (p means they are both pyranose rings).
  • Polysaccharide was dissolved, size reduced, chemically activated and buffer- exchanged by ultrafiltration.
  • Purified CRM 197 was then conjugated to the activated polysaccharide utilizing nickel chloride in the reaction mixture, and the resulting conjugate was purified by ultrafiltration prior to a final 0.2-micron filtration.
  • process parameters within each step such as pH, temperature, concentration, and time were controlled to yield conjugates with desired attributes.
  • Purified pneumococcal capsular polysaccharide powder was dissolved in water, and 0.45-micron filtered. Dissolved polysaccharide was homogenized to reduce the molecular mass. Homogenization pressure and number of passes through the homogenizer were controlled to 200 bar/5 passes followed by 500 bar/5 passes to size-reduce to a target molecular mass. Size- reduced polysaccharide was then concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
  • the polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.
  • Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.
  • the amount of sodium metaperiodate added was 0.15 moles of sodium metaperiodate per mole of polysaccharide repeating unit to achieve a target level of polysaccharide activation (moles aldehyde per mole of polysaccharide repeating unit).
  • the oxidation reaction proceeded for 2 hours at 22°C.
  • the activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was conducted at 2-8°C.
  • Oxidized polysaccharide solution was mixed with water and 1.5 M potassium phosphate pH 7.0.
  • the buffer pH selected was to improve the stability of activated
  • the batch was diluted to a polysaccharide concentration of approximately 3.0 g/L, cooled to 2-8°C, and 1.2-micron filtered.
  • the batch was diafiltered against 100 mM potassium phosphate, pH 7.0 at 2-8°C using a 100 kDa NMWCO tangential flow ultrafiltration membrane.
  • the batch, recovered in the retentate, was then diluted to approximately 2.0 g polysaccharide/L and pH-adjusted with the addition of 1.2 M sodium bicarbonate, pH 9.4.
  • Sodium borohydride (1 mole per mole of polysaccharide repeating unit) was added. 1.5 M potassium phosphate, pH 6.0 was later added.
  • the batch was then concentrated and diaftiltered against 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane. Polysorbate 20 was added to the retentate batch to a concentration of 0.05% (w/v) then the batch was 0.2 micron filtered.
  • the batch was adjusted to a polysaccharide concentration of 1.0 g/L with additional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 buffer with 0.03% (w/v) polysorbate 20.
  • the batch was dispensed into aliquots and frozen at ⁇ -60°C.
  • Table 1 shows the attributes of S. pneumoniae serotype 16F conjugate prepared according to the methods described above under aqueous conditions.
  • Activation sites on the S. pneumoniae serotype 16F polysaccharide were identified by reacting the (hydrated) aldehydes with thiosemicarbazide (TSC) in 5 mM citrate buffer at pH 5. TSC reacts with aldehydes (as well as hydrated aldehydes) to form an imine (secondary aldimine). The imine proton formed has a unique chemical shift that is downfield of the polysaccharide signals and was used to probe the oxidation sites of the polysaccharide.
  • TSC thiosemicarbazide
  • Oxidized serotype 16F polysaccharide (prepared as described in Example 3) was diluted with sodium citrate buffer pH 5 to a concentration of 10 mM then reacted with thiosemicarbazide (60 mM final concentration), mixed continuously at ambient temperature for 24 hours, and then lyophilized. The lyophilized sample was dissolved with 0.9 mL deuterium oxide for NMR analysis.
  • NMR experiments were carried out at 600 MHz at probe temperature of 27°C using a cryogenically cooled probe.
  • a ID proton spectrum was acquired using a 90 degree pulse with 16 transients and a 10 second delay between pulses (including 3 seconds of acquisition time).
  • TOCSY and Gradient COSY data were acquired with 4 transients in the first dimension and 256 and 512 increments in the second dimension respectively.
  • a multiplicity edited gHSQCAD was acquired with 32 transients and 512 increments.
  • the ID proton spectrum showed the imine signal as overlapping triplets of similar line shape and peak height at ⁇ 7.48 and 7.49 ppm (Figure 2B).
  • gCOSY and TOCSY data show cross peaks from the imine signals to signals centered on 4.57 ppm. From the gHSQCAD data, three signals are observed at proton chemical shift ⁇ 4.57 ppm but different carbon chemical shifts ( Figures 6 and 7).
  • the downfield signal at ⁇ 107 ppm (carbon) is the anomeric carbon of ⁇ -glucose.
  • the other signals at 67.16 and 67.41 ppm were identified as methylenes (methylene carbons have opposite phase of methine and methyl carbons in the multiplicity edited gHSQCAD experiment).
  • EXAMPLE 5 Conjugation of S. pneumoniae Serotype 16F Polysaccharide to CRM197 using Reductive Amidation in Dimethyl Sulfoxide Polysaccharide was dissolved, sized to a target molecular mass, chemically activated and buffer-exchanged by ultrafiltration. Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in dimethylsul oxide (DMSO).
  • DMSO dimethylsul oxide
  • Purified pneumococcal capsular Ps powder was dissolved in water and 0.45- micron filtered. Dissolved polysaccharide was homogenized to size reduce the molecular mass of the Ps. Homogenization pressure and number of passes through the homogenizer were controlled to 800-1000 bar/5 passes.
  • Size-reduced polysaccharide was concentrated and diafiltered against water using a 5 or 10 kDa NMWCO tangential flow ultrafiltration membrane.
  • the polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.
  • Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.
  • the amount of sodium metaperiodate added was 0.18 moles of sodium metaperiodate per mole of polysaccharide repeating unit to achieve a target level of polysaccharide activation (moles aldehyde per mole of polysaccharide repeating unit).
  • the oxidation reaction proceeded for 2 hours at 22°C.
  • the activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 5 or 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was conducted at 2-8°C.
  • Purified CRM197 obtained through expression in Pseudomonas fluorescens as previously described (WO 2012/173876 Al), was diafiltered against 2 mM phosphate, pH 7.0 buffer using a 5 kDa NMWCO tangential flow ultrafiltration membrane and 0.2-micron filtered.
  • Activated polysaccharides were formulated for lyophilization at 6 mg Ps/mL with sucrose concentration of 5% w/v.
  • CRM197 was formulated for lyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.
  • Formulated Ps and CRM197 solutions were individually lyophilized. Lyophilized Ps and CRM197 materials were redissolved individually in equal volumes of DMSO. The polysaccharide solution was spiked with sodium chloride to a concentration of 25-50 mM. The polysaccharide and CRM197 solutions were blended to achieve a polysaccharide concentration of 1.5-2.0 g Ps/L (grams polysaccharide/liter) and a polysaccharide to CRM197 mass ratio of 1.5.
  • the mass ratio was selected to control the polysaccharide to CRM197 ratio in the resulting conjugate.
  • Sodium cyanoborohydride (1 mole per mole of polysaccharide repeating unit) was added, and conjugation proceeded for 2 hours at 22°C. Reduction with sodium borohydride
  • the batch was then concentrated and diaftiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane.
  • the retentate batch was 0.2 micron filtered then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots and frozen at ⁇ -60°C.
  • Table 2 shows the attributes of S. pneumoniae serotype 16F conjugate prepared according to the methods described above in DMSO. Table 2. Attributes of S. pneumoniae serotype 16F conjugate from DMSO conjugation
  • Pneumococcal polysaccharide-CRM197 conjugates were prepared as described in
  • Example 3 The required volume of bulk conjugates needed to obtain the target concentration of invidual serotypes were calculated based on batch volume and concentration of individual bulk polysaccharide concentrations. Individual serotypes were combined with excipients, sterile filtered and added to APA under mixing conditions. The final concentration of each monovalent conjugate vaccine was 4 ⁇ g/mL (w/v PnPs) with 20 mM Histidine, 150 mM NaCl, 0.2% (w/v) PS-20 and 0.250 mg/mL (w/v Al) in the form of APA.
  • the immacheicity of the monovalent conjugates was evaluated in a New Zealand White Rabbit (NZWR) model.
  • NZWR New Zealand White Rabbit
  • IM intramuscularly
  • Monovalent pneumococcal vaccine was dosed at 1 ⁇ g PnPs (S. pneumoniae serotype 16F polysaccharide conjugated to CRM197) with 62.5 ⁇ g aluminum phosphate adjuvant (APA) per immunization.
  • Sera were collected prior to study start (pre-immune) and on days 14 (post-dose 1, PD1) and 28 (post-dose 2, PD2).
  • NZWRs were observed at least daily by trained animal care staff for any signs of illness or distress.
  • the vaccine formulations in NZWRs were deemed to be safe and well tolerated. All animal experiments were performed in strict accordance with the recommendations in the Guide for Care and Use of Laboratory Animals of the National Institutes of Health.
  • the NZWR experimental protocol was approved by the Institutional Animal Care and Use Committees at both Merck & Co., Inc (Kenilworth, NJ) and Covance (Denver, PA).
  • NZWR sera were tested in ELISA assays to evaluate IgG immunogenicity using a 1-2 mg/ml respective PnPs coating concentration. Functional antibody was determined through opsonophagocytosis assays (OP A) based on previously described protocols. See, e.g., Caro- Aguilar et al., 2017, Vaccine 35:865-72 and Burton et al., 2006, Clin Vaccine Immunol 13(9): 1004-9.
  • OP A ops
  • a multivalent pneumococcal conjugate vaccine consisting of different conjugate bulk blend preparations (including from S. pneumoniae serotypes 16F, 23A, 23B, 24F and 31) was prepared using pneumococcal polysaccharide-CRM197 conjugates and was formulated in 20 mM histidine pH 5.8 and 150 mM sodium chloride and 0.1% w/v polysorbate-20 (PS-20) at 4 ⁇ g/mL each serotype for a total polysaccharide concentration of 84 ⁇ g/mL.
  • the conjugates were prepared by individually conjugating the CRM197 protein to pneumococcal polysaccharide (PnPs) types (including from S.
  • the required volume of bulk conjugates needed to obtain the target concentration of individual serotypes was calculated based on batch volume and concentration of individual bulk polysaccharide concentrations.
  • the individual conjugates were added to a solution of histidine, sodium chloride and Polysorbate-20 (PS-20) to create the conjugate blend.
  • the formulation vessel containing the conjugate blend was mixed using a magnetic stir bar, and sterile filtered into another vessel. The formulations were then filled into plastic syringes, glass syringes, or vials and stored at 2-8°C.
  • Example 8 on day 0 and day 14 (alternating sides).
  • the multivalent pneumococcal conjugate vaccine was dosed at 2 ⁇ g of each conjugated PnPs per immunization.
  • Sera were collected prior to study start (pre-immune) and on days 14 (post-dose 1, PD1) and 28 (post-dose 2, PD2).
  • NZWRs were observed at least daily by trained animal care staff for any signs of illness or distress.
  • the vaccine formulations in NZWRs were deemed to be safe and well tolerated. All animal experiments were performed in strict accordance with the recommendations in the Guide for Care and Use of Laboratory Animals of the National Institutes of Health.
  • the NZWR experimental protocol was approved by the Institutional Animal Care and Use Committees at both Merck & Co., Inc and Covance (Denver, PA).
  • NZWR sera were evaluated for IgG immunogemcity using a multiplexed electrochemiluminescence (ECL) assay.
  • ECL electrochemiluminescence
  • This assay was developed for use with rabbit serum based on the human assay described by Marchese et al. (Optimization and validation of a multiplex, electrochemiluminescence-based detection assay for the quantitation of
  • Polysaccharide-protein conjugates prepared from S. pneumoniae serotypes 16F, 23 A, 23B, 24F, and 31 in a multivalent pneumococcal conjugate vaccine were found to be immunogenic for both post dose 1 (PD1) and post dose 2 (PD2) in rabbits ( Figure 11). They also generated functional antibody which killed vaccine-type bacterial strains ( Figure 12). Rabbits immunized with the multivalent pneumococcal conjugate vaccine at the 2 ⁇ g dose had significantly higher PD1 MOPA titers for four serotypes compared to pre-immune rabbit sera (Figure 12).

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Abstract

La présente invention concerne des polysaccharides capsulaires en provenance de sérotypes de Streptococcus pneumoniae identifiés par RMN. La présente invention concerne en outre des conjugués polysaccharide-protéine dans lesquels des polysaccharides capsulaires en provenance d'un ou plusieurs de ces sérotypes sont conjugués à une protéine porteuse telle que CRM197. Des conjugués polysaccharide-protéine en provenance d'un ou plusieurs de ces sérotypes peuvent être inclus dans des vaccins conjugués antipneumococciques multivalents comprenant des polysaccharides en provenance de multiples autres sérotypes de Steptococcus pneumoniae.
PCT/US2018/049305 2017-09-07 2018-09-04 Polysaccharides antipneumococciques et leur utilisation dans des conjugués immunogènes polysaccharide-protéine porteuse WO2019050813A1 (fr)

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RU2020112309A RU2801288C2 (ru) 2017-09-07 2018-09-04 Пневмококковые полисахариды и их применение в иммуногенных конъюгатах полисахарид-белок-носитель
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US16/645,023 US11395849B2 (en) 2017-09-07 2018-09-04 Pneumococcal polysaccharides and their use in immunogenic polysaccharide-carrier protein conjugates
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BR112020004471A BR112020004471A8 (pt) 2017-09-07 2018-09-04 Polissacarídeos pneumocócicos e uso dos mesmos em conjugados imunogênicos polissacarídeo-proteína carreadora
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